Fermentation process for antibody production

ABSTRACT

A feedback control mechanism for a fermentation of yeast cells to make recombinant proteins uses a respiratory quotient measurement which adjusts the levels of oxygenation and/or fermentable sugar feed. The feedback control mechanism permits well controlled cultures that produce good amounts of product while avoiding toxic accumulation of ethanol. Additionally, recombinant proteins so produced have excellent qualitative properties, such as excellent homogeneity and proper inter-subunit assembly.

RELATED APPLICATIONS

This application is a U.S. National Phase Application submitted under 35U.S.C. 371 based on International Application No. PCT/US14/30311 filedMar. 17, 2014 (published as WO 2014-145521 on Sep. 18, 2014), whichclaims priority to and benefit of U.S. Provisional Application No.61/790,613 filed Mar. 15, 2013, each of which is hereby incorporated byreference in its entirety.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of fermentation. In particular, itrelates to fermentation of recombinant yeast cells.

BACKGROUND OF THE INVENTION

The yeast Pichia pastoris has previously is a suitable production hostfor the manufacture of recombinant proteins of therapeutic utility.Examples include the production of Human Serum Albumin and theKallikrein inhibitor, Ecallantide (Reichert, J. mAbs 4:3 1-3, 2012).Pichia pastoris has been used for the production of recombinantmonoclonal antibodies having correctly assembled heavy and light chains(U.S. Pat. No. 7,927,863). The glyceraldehyde-3-phosphate dehydrogenase(GAP) promoter can drive expression in yeast of an antibody lackingN-glycosylation in yeast (U.S. Pat. No. 7,927,863). Recent work byBaumann et. al. (BMC Genomics 2011, 12:218), using the GAP system toproduce recombinant antibody Fab fragment has shown that this system,previously thought to be constitutive, exhibits increased expressionunder hypoxic conditions using glucose as the source of carbon andenergy. Hypoxic conditions are those that allow the dissolved oxygenlevel in a fermentation to drop to very low levels while still supplyingoxygen to the culture through aeration and agitation. This results inmixed aerobic and fermentative metabolism.

The use of hypoxic conditions in a fermentor can result in the toxicaccumulation of ethanol, and care must be exercised to control theprocess such that toxic levels do not accumulate. Baumann accomplishedthis by measuring the level of ethanol in the fermentor and adjustingthe glucose feed rate to reduce its accumulation. However this method isnot very scalable, as technology for reliably measuring ethanol in largescale fermentors is not widely available.

There is a continuing need in the art for efficient and accurateproduction of recombinant proteins, especially antibody molecules,antibody fragments, and antibody constructs.

SUMMARY OF THE INVENTION

According to one embodiment of the invention a method is provided thatproduces a recombinant protein such as an antibody or antigen-bindingfragment of an antibody in yeast cells. A population of yeast cells iscultured under fed-batch fermentation conditions in a culture medium.Each yeast cell comprises a DNA segment encoding a recombinant protein,such as a heavy chain polypeptide, and optionally a DNA segment encodinga second recombinant protein, such as a light chain polypeptide of anantibody. The one or more DNA segments are operably linked to aglyceraldehyde-3-phosphate (GAP) transcription promoter and atranscription terminator. The fermentation comprises a fermentable sugarfeed at a first feed rate, and the fermentation is oxygenated at a firstoxygen transfer rate. The respiratory quotient (RQ) of the population ismeasured during the feeding phase of the fed-batch fermentation and itis compared to a desired predetermined range. One or both of thefermentable sugar feed rate and the oxygen transfer rate are adjusted toa second rate when the RQ is outside of a desired predetermined range.The measuring and adjusting are performed throughout all or part of theculturing. The yeast cells are harvested from the culture medium.Recombinant proteins, such as heavy chain and light chain polypeptidesproduced by the yeast cells, are recovered from yeast cell-depletedculture medium or from the yeast cells.

According to another embodiment of the invention a method is providedfor producing an antibody comprising two heavy chains and two lightchains in Pichia yeast cells. A population of Pichia yeast cells iscultured under hypoxic, fed-batch fermentation conditions in a culturemedium. Each yeast cell comprises a DNA segment encoding a heavy chainpolypeptide and a DNA segment encoding a light chain polypeptide of anantibody. DNA segments are operably linked to aglyceraldehyde-3-phosphate (GAP) transcription promoter and atranscription terminator. The yeast cells are harvested from the culturemedium. The antibody produced by the yeast cells is recovered from theyeast cells or from the yeast cell-depleted culture medium.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification, provide the art with methods andproducts with improved reliability, predictability, efficiency, andquality.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows the RQ profiles of 3 different Mab1 Strains A, B, and C.The vertical line indicates the time at which RQ control was initiated.

FIG. 2: shows the Agitation profiles of 3 different Mab1 Strains A, B,and C. The vertical line indicates the time at which RQ control wasinitiated.

FIG. 3: shows the Ethanol profiles of 3 different Mab1 Strains A, B, andC.

FIG. 4: shows the Growth profiles of 3 different Mab1 Strains A, B, andC.

FIG. 5: shows the Whole Broth Titer profiles of 3 different Mab1 StrainsA, B, and C.

FIG. 6: shows the RQ profiles of Mab1 Strain A at 3 different RQ Setpoints RQ 1.09-1.15, 1.19-1.25, 1.29-1.35. The vertical line indicatesthe time at which RQ control was initiated.

FIG. 7: shows the Agitation profiles of Mab1 Strain A at 3 different RQSet points RQ 1.09-1.15, 1.19-1.25, 1.29-1.35. The vertical lineindicates the time at which RQ control was initiated.

FIG. 8: shows the Ethanol profiles of Mab1 Strain A at 3 different RQSet points RQ 1.09-1.15, 1.19-1.25, 1.29-1.35

FIG. 9: shows the Growth profiles of Mab1 Strain A at 3 different RQ Setpoints RQ 1.09-1.15, 1.19-1.25, 1.29-1.35

FIG. 10: shows the Whole Broth Titer profiles of Mab1 Strain A at 3different RQ Set points RQ 1.09-1.15, 1.19-1.25, 1.29-1.35

FIG. 11: shows the RQ profiles of Mab1 Strain A grown under aerobic andhypoxic conditions. The vertical line indicates the time at which RQcontrol was initiated.

FIG. 12: shows the Agitation profiles of Mab1 Strain A grown underaerobic and hypoxic conditions. The vertical line indicates the time atwhich RQ control was initiated.

FIG. 13: shows the Ethanol profiles of Mab1 Strain A grown under aerobicand hypoxic conditions

FIG. 14: shows the Growth profiles of Mab1 Strain A grown under aerobicand hypoxic conditions

FIG. 15: shows the % DO profiles of Mab1 Strain A grown under aerobicand hypoxic conditions

FIG. 16: shows the Whole Broth Titer profiles of Mab1 Strain A grownunder aerobic and hypoxic conditions

FIG. 17: shows the RQ profiles of 4 different Mab2 Strains A, B, C andD. The vertical line indicates the time at which RQ control wasinitiated.

FIG. 18: shows the Agitation profiles of 4 different Mab2 Strains A, B,C and D strains. The vertical line indicates the time at which RQcontrol was initiated.

FIG. 19: shows the Ethanol profiles of 4 different Mab2 Strains A, B, Cand D strains.

FIG. 20: shows the Growth profiles of 4 different Mab2 Strains A, B, Cand D strains.

FIG. 21: shows the Whole Broth Titer profiles of 4 different Strains A,B, C and D strains.

FIG. 22: shows the RQ profiles of 3 different Mab3 Strains A, B, and C.The vertical line indicates the time at which RQ control was initiated.

FIG. 23: shows the Agitation profiles of 3 different Mab3 Strains A, B,and C. The vertical line indicates the time at which RQ control wasinitiated.

FIG. 24: shows the Ethanol profiles of 3 different Mab3 Strains A, B,and C.

FIG. 25: shows the Growth profiles of 3 different Mab3 Strains A, B, andC.

FIG. 26: shows the Whole Broth Titer profiles of 3 different Mab3Strains A, B, and C.

FIG. 27: shows the RQ profiles of Mab3 Strain B at different feed rates.The vertical line indicates the time at which RQ control was initiated.

FIG. 28: shows the Agitation profiles of Mab3 Strain B at different feedrates. The vertical line indicates the time at which RQ control wasinitiated.

FIG. 29: shows the Ethanol profiles of Mab3 Strain B at different feedrates.

FIG. 30: shows the Growth profiles of Mab3 Strain B at different feedrates.

FIG. 31: shows the Whole Broth Titer profiles of Mab3 Strain B atdifferent feed rates.

FIG. 32: shows the RQ profiles of Mab4 Strain A at different feed rates.The vertical line indicates the time at which RQ control was initiated.

FIG. 33: shows the Agitation profiles of Mab4 Strain A at different feedrates. The vertical line indicates the time at which RQ control wasinitiated.

FIG. 34: shows the Ethanol profiles of Mab4 Strain A strain at differentfeed rates.

FIG. 35: shows the Growth profiles of Mab4 Strain A strain at differentfeed rates.

FIG. 36: shows the Whole Broth profiles of Mab4 Strain A strain atdifferent feed rates.

FIG. 37: shows the Whole Broth profiles of Mab2 Strain A for the aerobic(B1) and hypoxic (B2) process in large scale fermentation

FIG. 38: shows the SDS-PAGE Non-Reduced and Reduced gels of Mab1 StrainA at fermentation times 62, 70, and 86 hours for aerobic and hypoxicprocess conditions

FIG. 39: shows the SDS-PAGE Non-Reduced and Reduced gels of Mab1 StrainA at fermentation times 62, 69, and 85 hours

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed a process for making recombinant proteinsin yeast which may employ a particular type of feedback controlmechanism to increase the productivity of fermentations. That feedbackcontrol mechanism allows the robust and precise control of mixed aerobicand fermentative metabolism that stimulates optimal production of thedesired product. This can be used to good effect to produce recombinantmonoclonal proteins such as antibodies in yeast, particularly in Pichiapastoris, and more particularly using the glyceraldehyde-3-phosphate(GAP) promoter.

The process using the feedback control mechanism is applicable to theproduction of full-length, correctly assembled recombinant monoclonalantibodies, as well as to antibody fragments and other recombinantproteins, i.e., not glyceraldehyde-3-phosphate. The control mechanismthat we employ is easy to mechanize and render automatic, thuseliminating much labor in monitoring and adjusting fermentationconditions. The process is applicable to production of a variety ofantibodies and other recombinant proteins and is readily scalable toaccommodate commercial, e.g., large scale, production needs.

The production process may use Respiratory Quotient (RQ) as a feedbackcontrol variable. RQ can be used to balance mass transfer parametersand/or fermentable sugar feed rate in order to maintain a hypoxic statein the culture while preventing the toxic accumulation of ethanol, aby-product of fermentative metabolism. RQ is defined as the molar rateof carbon dioxide produced divided by the molar rate of oxygen consumedin the culture. It can be measured by analyzing the exhaust gas comingfrom the fermentor for content of carbon dioxide and oxygen. Thismetabolic parameter can be measured continuously or intermittentlythroughout the desired growth phase using readily available means.Examples of appropriate intervals for measurements are hourly,half-hourly, quarter-hourly, ten minutes, five minutes, four minutes,three minutes, two minutes, one minute. Time periods during measurementsmay vary with growth conditions, from initiating the culture throughharvest. Exemplary periods for measurement and control are between 20and 40 hours, between 10 and 60 hours, between 5 and 70 hours, andbetween 20 and 110 hours after initiating of the culturing in thefermentor.

When yeast cells are grown in a completely anaerobic state, without thepresence of oxygen, they are said to be using fermentative metabolism toproduce the energy they need to grow. In this case the followingstoichiometric equation for the conversion of glucose to ethanolapplies:

C₆H₁₂O₆→2C₂H₅OH+2CO₂+H₂O+Energy

When yeast cells obtain their energy solely from aerobic metabolism ofglucose, then oxygen is consumed, and only carbon dioxide and water areproduced:

C₆H₁₂O₆+3O₂→3CO₂+6H₂O+Energy

In the presence of oxygen, yeast cells use aerobic metabolism, which ismore efficient, i.e., more energy is obtained from a mole of glucoseunder aerobic metabolism than under fermentative metabolism.

The RQ of a culture producing only ethanol from glucose approachesinfinity (since no oxygen is consumed, the denominator of RQ is zero),whereas for purely aerobic metabolism of glucose the RQ approaches thevalue of 1.0 (three moles of oxygen are consumed to produce 3 moles ofcarbon dioxide). Thus values higher than 1 indicate a mixed metaboliccondition where both aerobic and fermentative metabolism arc takingplace simultaneously. Typically oxygen transfer rate and/or fermentablesugar feed rate can be adjusted using RQ as a feedback control variableto accomplish this mixed metabolism. Using such a mixed metabolism,hypoxic conditions can be maintained. A hypoxic state exists when thereis a low level of fermentative metabolism controlled by the equilibriumof oxygen transfer rate and fermentable sugar feed rate. Hypoxicconditions may be defined by an RQ above 1.0 with dissolved oxygen belowabout 5%.

RQ can be measured in the exhaust gas stream from a fermentor. Any knownand suitable method for ascertaining the molar concentration of oxygenconsumed and carbon dioxide generated can be used. Exemplary techniqueswhich may be used are mass spectrometry, infrared spectroscopy, andparamagnetic analysis.

Hypoxic growth has a beneficial effect on the production of full length,properly assembled proteins, such as antibodies, in Pichia. We tried toreduce the dissolved oxygen concentration simply by reduction of theagitator speed during fermentation. However, it was not possible toobtain reliable control in this manner, because small differences inagitation rate or fermentable sugar feed rate would quickly result inthe accumulation of toxic levels of ethanol.

A feedback control mechanism can also be used to measure and controlethanol levels through modulation of either fermentable sugar feed rateand/or oxygen transfer rate, e.g., by agitator speed. Controllingaccumulation of ethanol should permit a more stable process. In order tomonitor ethanol levels one can use a probe inserted into the fermentor.The probe can monitor ethanol levels in the fermentation brothcontinuously. However, it is not feasible to use such a probe incommercial manufacturing of molecules under Good ManufacturingProcesses, because it does not have an output that can be sufficientlycalibrated.

When RQ is maintained in a narrow range from approximately 1.1 toapproximately 2, ethanol accumulation stabilizes at levels that are nottoxic. Preferably the concentration of ethanol is maintained betweenabout 5 g/l and 17 g/l. Moreover, these same conditions stimulate theGAP promoter, leading to significantly increased recombinant protein,e.g., antibody production over aerobic fermentation conditions. RQranges that may be desirable include about 1.08-2.0; about 1.08-1.85;about 1.08-1.65; about 1.08-1.45; about 1.08-1.35; about 1.08-1.25;about 1.08-1.2; and about 1.08-1.15. Alternative carbon sources otherthan glucose can achieve an RQ less than 1. Such carbon sources includeacetate and glycerol. Other suitable RQ ranges include 1.08 to 1.35, and1.15 to 1.25. RQ can be monitored and controlled during any desiredportion of the fermentation, for example from 0 to 110 hours, from 20-40hours, from 20-70 hours, from 20-90 hours, from 20-110 hours, or anyother desired time period.

Thus RQ can be manipulated and changed over time by addition of variouscarbon sources, by addition of various amounts of a carbon source, andby manipulation of the oxygen levels. In one embodiment, oxygen levelsare manipulated by increasing or decreasing agitation. In anotherembodiment, the ratio of oxygen to nitrogen gas in the gas feed iscontrolled. Ways that the oxygen transfer rate can be adjusted includethe changing the air flow rate, the oxygen concentration, the celldensity, the temperature, and agitation. In another embodiment glucoseor other fermentable sugar feed is modulated to affect the RQ. Otherfermentable sugars which can be used in the feed include withoutlimitation fructose, sucrose, maltose, and maltotriose. Feed rate orcomposition can be modulated to affect the RQ. The control of RQ may bemanual or automatic.

Protein-encoding nucleic acids, e.g., encoding antibodies, may be on asingle or multiple continuous or discontinuous segments of a recombinantconstruct. Antibodies may be any type of fragment or construct or fulllength. These may be, for example, Fab, F(ab′)₂, Fc, and ScFv. In someembodiments, the chains and or chain fragments will assemble properly invivo. If assembly is not proper, in vitro assembly may be necessary.Other proteins which may be desirably made are those having one or moresubunits, whether heteromeric or homomeric. Typically the protein willbe useful for diagnostic or therapeutic purposes. The protein may be agrowth factor, a cytokine, a blood coagulation factor, a therapeutictoxin, a structural protein useful for reconstruction, an enzyme, etc.

Proteins such as antibodies may be recovered from the cell-depletedculture medium or from the cells by any technique known in the art.Typically a binding step will be used to reduce the volume of thepreparation. Binding can be done on filters or columns or other solidsupports, as is convenient. In some embodiments, protein A may be usedas an antibody capturing agent. The protein A may be bound to apolymeric matrix.

Any type of yeast cells can be used, including Saccharomyces, Hansenula,and Pichia species. Exemplary but not limiting species which may be usedare P. pastoris, P. methanolica, P. angusta, P. thermomethanolica,Hansenula polymorpha, and S. cerevisiae. The yeast may be haploid ordiploid.

Other promoters like GAP may be used similarly. These are typicallypromoters that are for genes that are up-regulated in hypoxic,glucose-limiting growth in yeast cells, such as Pichia. Such promoterswhich may be used include, without limitation, promoters for genesYHR140W, YNL040W, NTA1, SGT1, URK1, PGI1, YHR112C, CPS1, PET18, TPA1,PFK1, SCS7, YIL166C, PFK2, HSP12, ERO1, ERG11, ENO1, SSP120, BNA1, DUG3,CYS4, YEL047C, CDC19, BNA2, TDH3, ERG28, TSA1, LCB5, PLB3, MUP3, ERV14,PDX3, NCP1, TPO4, CUS1, COX15, YBR096W, DOG1, YDL124W, YMR244W, YNL134C,YEL023C, PIC2, GLK1, ALD5, YPR098C, ERG1, HEM13, YNL200C, DBP3, HAC1,UGA2, PGK1, YBR056W, GEF1, MTD1, PDR16, HXT6, AQR1, YPL225W, CYS3, GPM1,THI11, UBA4, EXG1, DGK1, HEM14, SCO1, MAK3, ZRT1, YPL260W, RSB1, AIM19,YET3, YCR061W, EHT1, BAT1, YLR126C, MAE1, PGC1, YHL008C, NCE103, MIH1,ROD1, FBA1, SSA4, PIL1, PDC1-3, THI3, SAM2, EFT2, and INO1.

Large scale fermentation processes are those typically used incommercial processes to produce a useful product. Typically these aregreater than 100 liters in volume. Fed-batch fermentation is a processby which nutrients are added during the fermentation to affect celldensity and product accumulation.

Disclosures of prior published patent applications and patents, U.S.Pat. Nos. 7,927,863, 8,268,582, U.S. App 2012/0277408 are expresslyincorporated herein.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLES

We show the applicability of the method to the production of fourdifferent humanized monoclonal antibodies. Each antibody is produced inPichia pastoris using the glyceraldehyde-3-phosphate (GAP) promotersystem.

Example 1

We found a difference in titers between aerobic and hypoxic cultures ofantibody Mab2. Restricting the oxygen availability to the culture byreducing the agitation rate in the fermentor resulted in a significantincrease in product formation. This was our first confirmation thathypoxic conditions, when applied to the production of full lengthantibodies, results in a significant increase in product formation forfully assembled, appropriately disulfide bonded humanized monoclonalantibodies. See FIG. 37.

Example 2

Three different strains of antibody Mab1, each with differing copynumbers, are grown in 20 liter fermentors using RQ control strategy(modulaton of agitation using feedback control that modulates agitatorspeed to maintain RQ at the desired level (in this case a value of 1.12)to promote mixed metabolism of hypoxic conditions in a controlled mannerso as to ensure that ethanol concentrations do not reach toxic levels.In each case, the robust nature of the feedback control mechanism allowsmixed metabolism without accumulation of toxic levels of ethanol(typically greater than 20 g/L). (See FIGS. 1-5)

Example 3

Mab1 is cultured under hypoxic condition using the RQ control strategy,at three different control set-points for RQ. In this case, increasingthe RQ set-point increases the level of ethanol accumulation, reducesthe accumulation of cells, but does not have a significant impact on theoverall product accumulation. This shows the utility of the RQ method atset-points ranging from 1.09 to 1.35. (See FIGS. 6-10)

Example 4

We compared, for Mab1, the effect of hypoxic growth, as attained by RQcontrol, against the same process under aerobic conditions. The aerobicprocess results in lower ethanol production (as expected), and markedlylower product formation. (See FIGS. 11-16).

Example 5

The RQ control strategy was implemented on fermentations of Mab2 thathad production strains varying in the number of copies of each heavy andlight chain. This study shows the robust nature of the RQ strategy incontrolling the accumulation of ethanol while providing a hypoxicenvironment for mixed metabolism. (See FIGS. 17-21).

Example 6

The RQ control strategy was implemented on fermentations of Mab3, thathad production strains varying in the number of copies of each heavy andlight chain. This study shows the robust nature of the RQ strategy incontrolling the accumulation of ethanol while providing a hypoxicenvironment for mixed metabolism. (See FIGS. 22-26)

Example 7

Strains of Mab3, containing varying copies of Heavy Chain and of LightChain is grown using the RQ control strategy but incorporating varyingglucose feed rates. Again, the RQ strategy allows for effective controlof ethanol levels, resulting in very similar product accumulation rates.This provides further evidence of the robustness of the RQ strategy to avariety of fermentation conditions. (See FIGS. 27-31).

Example 8

The RQ strategy is demonstrated for MAb4, which binds to the same targetas MAb1, but has a different sequence in its CDR than MAb1. We comparedtwo different rates of glucose feed. Once again the strategy allowed fora stable ethanol concentration and similar antibody accumulation rates.(See FIGS. 32-36).

Example 9

The Fermentation Process for the Production of Antibodies orAntigen-Binding

Fermentation Media

Inoculum Medium is described below in Table 1.

TABLE 1 Inoculum Medium Component¹ Final concentration Yeast extract  30 g/l KH₂PO₄ 27.2 g/l Glycerol or Glucose   20 g/l Yeast nitrogenbase w/o amino acids 13.4 g/l Biotin  0.4 mg/l ¹Keeping the samemolarity, any chemical (X nH₂O; n ≥ 0) can be replaced by anotherchemical containing the same activated ingredient but various amount ofwater (X kH₂O; k ≠ n).

Seed Fermentation Medium

Medium is described below in Table 2.

Composition Seed Fermentation Medium Component¹ Final concentrationSodium citrate dihydrate 10.0 g/l MgSO₄—7H₂O  3.7 g/l NH₄H₂PO₄ 36.4 g/lK₂HPO₄ 12.8 g/l K₂SO₄ 18.2 g/l Glycerol, anhydrous 40.0 g/l Yeastextract 30.0 g/l Antifoam 204  0.5 ml/l Trace mineral solution (PTM1)4.35 ml/l ¹Keeping the same molarity, any chemical (X nH₂O; n ≥ 0) canbe replaced by another chemical containing the same activated ingredientbut various amount of water (X kH₂O; k ≠ n).Trace Mineral Solution is described below in Table 3.

TABLE 3 Trace Mineral Solution (PTM1) Component¹ Final concentrationZnCl₂ ¹ or Zinc Sulphate Heptahydrate² 20 g/l¹ or 35 g/l² FeSO₄—7H₂O  65 g/l 95-98% H₂SO₄   5 ml/l NaI 0.08 g/l MnSO₄—2H₂O   3 g/lNa₂MoO₄—2H₂O  0.2 g/l H₃BO₃ 0.02 g/l CoCl₂  0.5 g/l CuSO₄—5H₂O   6 g/lBiotin  0.2 g/l ¹Keeping the same molarity, any chemical (X nH₂O; n ≥ 0)can be replaced by another chemical containing the same activatedingredient but various amount of water (X kH₂O; k ≠ n).

When all components are completely dissolved in DI water, filtersterilize through a sterile 0.2 μm filter.

Production Culture Batch Medium is described below in Table 4

TABLE 4 Production Culture Batch Medium Component1 Final concentrationSodium citrate dihydrate 10.0 g/l MgSO4—7H2O  3.7 g/l NH4H2PO4 35.6 g/lK2HPO4 12.8 g/l K2SO4 18.2 g/l Glycerol, anhydrous 40.0 g/l Yeastextract 30.0 g/l Antifoam 204  1.6 ml/l 1Keeping the same molarity, anychemical (X nH2O; n ≥ 0) can be replaced by another chemical containingthe same activated ingredient but various amount of water (X kH2O; k ≠n).

The above is sterilized by autoclaving at 121° C. for a minimum of 20minutes. After sterilization and cooling, 4.35 ml/l of trace mineralsolution (PTM1) is added to the Production Culture Batch Medium. Priorto inoculation of the fermentor, Production Culture Batch Mediumcontaining 4.35 ml/l of PTM1 should be adjusted to pH 6.0 with 24-30%NH₄OH. The above values should be based on the total fermentationstarting volume, including both medium and inoculum culture.

Glucose/Yeast Extract Feed Solution is described below in Table 5.

Component¹ Final concentration Dextrose, anhydrous  500 g/l Yeastextract   50 g/l MgSO₄—7H₂O   3 g/l Antifoam 204  0.1 ml/l Sodiumcitrate dihydrate 1.66 g/l PTM1   12 ml/l ¹Keeping the same molarity,any chemical (X nH₂O; n ≥ 0) can be replaced by another chemicalcontaining the same activated ingredient but various amount of water (XkH₂O; k ≠ n).Ethanol bolus composition is described below in Table 6.

Component¹ Final concentration Ethanol, 200 Proof 11 g/l ¹Optionally amore dilute solution of ethanol can be used to achieve the same finalconcentration.

Fermentation Process

The fermentation process for the production of antibodies orantigen-binding fragments is accomplished by yeast, such as P. pastoris.The fermentation is initiated from the thawing of a frozen vial of aworking cell bank. The thawed cells are then propagated in shake flasks.The culture from the shake flask is then used in the Inoculum Step,followed by a fed-batch process for the production of antibody.Optionally, the inoculum can be used to propagate cells in a seed batchfermentation, which can then be used to inoculate the productionfermentor.

1. Inoculum Step

Thawed cells of the working cell bank are transferred to a baffled shakeflask (1 to 4 baffles) that contains 8-20% of the working volumecapacity of the flask Inoculum Medium. Thawed working cell bank is addedat 0.1-1.0% of the volume of inoculum medium to the shake flask. Theinoculum culture is incubated at 29-31° C. at an agitation speed of220-260 rpm. The seed culture is harvested once reaching a cell densitycorrelated to the absorbance at 600 nm (OD₆₀₀) of 15-30 (optimally20-30). The culturing time is usually 20-26 hours (optimally 23-25hours).

2. Seed Fermentation Batch Fermentation (Optional)

Fermentor is inoculated with inoculum from “Inoculum Step”

Inoculum=0.3% of seed fermentor medium volume

Temp: 30° C.

% DO: 30%

pH: 6.0

Agitation: Cascade Strategy from 100-490 RPM

Airflow: 1 vvm ((volume of air/volume of starting fermentormedia)/minute) Pressure: 0.2 bar

Oxygen supplementation will occur when maximum agitation is reached witha corresponding decrease in airflow to maintain a constant vvm, tomaintain the desired % DO set point of 30%

Continue Monitoring for DO Spike.

When a DO Spike has occurred which is indicated by a decrease inAgitation and an increase in DO, denoting that the carbon source(glycerol or glucose) has been completely utilized and the measuredoptical density, OD₆₀₀, is greater than 20, transfer a volume of theseed batch fermentation or inoculum culture which is equal to 1.0-10% ofthe Production fermentor Starting Batch Volume.

3. Batch Culture Phase

The batch culture is initiated by inoculation of the fermentor with theseed culture and ended with the depletion of glycerol. The fermentorcontains prepared Production Culture Batch medium at 30-40% of maximumworking volume. The seed culture is used to create a 1-10% inoculumwithin the fermentor. The initial engineering parameters are set asfollows:

-   -   Temperature: 27-29° C.;    -   Agitation (P/V): 2-16 KW/m³    -   Headspace Pressure: 0.7-0.9 Bar    -   Air flow: 0.9-1.4 VVM (volumes of air per volume of culture per        minute, based on starting volume)    -   DO: no control    -   pH: 5.9 to 6.1, controlled by 24-30% NH₄OH

The starting agitation speed and airflow are kept constant during theBatch Culture Phase in order to meet the initial power per volume (P/V)and volume per volume per minute (VVM) requirements. Batch Culture Phaseis ended by starting feed when glycerol is depleted. The depletion ofglycerol is indicated the dissolved oxygen (DO) value spike. The DOspike is defined as when the value of the DO increases by greater than30% within a few minutes. Batch Culture Phase usually lasts 10-15 hours(optimally 11-13 hours).

4. Ethanol Bolus Addition (Optional)

Upon observation of the DO spike as mentioned above, 8-16 g/l ofEthanol, 200 Proof as a bolus is added into the fermentor. This usuallyoccurs within 12-14 hours of Batch Culture Phase.

5. Fed-Batch Culture Phase

Feed to the fermentor with Glucose/Yeast Extract Feed Solution isinitiated after the DO spike and after Ethanol Bolus Addition, around12-14 hours within Batch Culture Phase and continues to the end of thefermentation. The rate of Glucose/Yeast Extract Feed Solution feed isset to allow for 6-11 g of glucose/I of starting volume per hour. Thestart of Glucose/Yeast Extract Feed Solution begins Fed-batch CulturePhase.

6. RQ Control Start

Respiratory Quotient (RQ) Control begins 8 hours after Fed-batch CulturePhase start. The initial RQ set points arc in the range of 1.09 to 1.35.Agitation is used to control the RQ. Agitation is cascaded off of the RQcontrol set point. RQ control starts at approximately 20-22 hours fromthe onset of Batch Culture Phase and continues to the end of thefermentation. The duration of RQ control lasts approximately 60 to 90hours.

The agitation is adjusted in order to maintain a set level of RQ. The RQControl strategy is detailed as follows:

RQ Hi Control set point: 1.35

RQ Low Control set point: 1.08

Maximum agitator set point: 255-950 rpm

Minimum agitator set point: 150-300 rpm

Agitator step change (change at each Wait Time interval): 3-25 rpm

Wait Time (time between evaluations): 3-10 minutes

Ethanol/RQ Control Strategy

This strategy has been incorporated to ensure that the ethanolconcentration does not exceed a maximum value that can be toxic to thecells, and does not exceed a minimum value that could reduce productexpression.

Example 10

FIG. 38 show SDS-PAGE gels of Mab1 produced under both hypoxic andaerobic conditions. For the non-reduced gel, in addition to the mainband at 150 kD, additional bands below the main band indicate productheterogeneity with respect to the level of interchain disulfidebridging. These gels show that the level of heterogeneity is reduced bythe use of hypoxic conditions. The increased homogeneity of the fulllength, completely cross-linked product indicates increased purity,i.e., increased desired product relative to other proteins present.

FIG. 38 also shows the reduced SDS-PAGE gel for the same samples. Inthis case expected bands at 25 kD and 50 kD represent the heavy andlight chains of the antibody. The additional bands, particularly the oneabove the Heavy chain at approximately 55 kD represent the present ofvariant species of the antibody. These gels show a dramatic reduction inthe presence of this variant when the cells are grown under hypoxicconditions, as compared with the aerobic culture.

1. A method for producing a recombinant protein in yeast cells,comprising the steps of: a) culturing under fed-batch fermentationconditions a population of yeast cells in a culture medium, wherein eachyeast cell comprises a DNA segment encoding a polypeptide, wherein saidDNA segment is operably linked to a glyceraldehyde-3-phosphate (GAP)transcription promoter and a transcription terminator, wherein theprotein is not glyceraldehyde-3-phosphate, wherein the fermentationcomprises a fermentable sugar feed at a first feed rate and wherein thefermentation is agitated at a first oxygen transfer rate; b) measuringrespiratory quotient (RQ) of the population during the batchfermentation and determining if it is within a desired predeterminedrange, wherein the desired predetermined range of RQ at about 20-40hours after initiation of the culturing is between about 1.08 and about1.35; c) adjusting one or both of the fermentable sugar feed rate to asecond feed rate or the oxygen transfer rate to a second oxygen transferrate, when the RQ is outside of a desired predetermined range; d)repeating steps (b) and (c) one or more times throughout the step ofculturing; e) harvesting the yeast cells from the culture medium; and f)recovering the polypeptide from the cells and/or the culture medium. 2.The method of claim 1 wherein step (d) is performed at intervals ofbetween about 1 and 5 minutes, at intervals of about 3 minutes, or isperformed continuously.
 3. (canceled)
 4. (canceled)
 5. The method ofclaim 1 wherein at least one adjustment to feed rate is made in step(c), step (c) is performed automatically using a feedback controlmechanism linked to a device which measures the RQ.
 6. (canceled)
 7. Themethod of claim 1 wherein the yeast cells are from a species selectedfrom the group consisting of Pichia pastoris, Pichia methanolica, Pichiaangusta, Pichia thermomethanolica, and Saccharomyces cerevisiae.
 8. Themethod of claim 1 further comprising the step of: delivering ethanol tothe yeast cells at about 10 to 14 hours of the culturing to achieve alevel in the fermentation of about 8.0 to about 12.0 g/l ethanol.
 9. Themethod of claim 1 comprising one or more of the following: (i) at leastthe desired predetermined range of RQ at about 20-40 hours afterinitiation of the culturing is between about 1.09 and about 1.25, (ii)the step of harvesting is performed at about 80-110 hours after theinitiation of the culturing, (iii) ethanol concentration is measuredduring the step of culturing, and adjustments are made to stabilize theethanol concentration above about 5 g/l and below about 25 g/l, whereinsaid adjustments are made by adjusting one or both of the fermentablesugar feed rate to a third feed rate or the oxygen transfer rate to athird oxygen transfer rate, optionally to stabilize the ethanolconcentration above about 5 g/l and below about 17 g/l.
 10. (canceled)11. (canceled)
 12. (canceled)
 13. The method of claim 9 wherein thedesired predetermined range of RQ at 20-110 hours of the fermentation isselected from the group consisting of: about 1.08-1.1; about 1.08-1.15;about 1.08-1.2; about 1.08-1.25; about 1.08-1.3; and about 1.08-1.35.14. The method of claim 1 wherein step (b) of measuring is performed bysampling the exhaust gas of the fermentation, or step (b) of measuringis performed using a mass spectrometer, infrared analyzer, orparamagnetic analyzer.
 15. (canceled)
 16. The method of claim 1 whereinin step (c) the oxygen transfer rate is adjusted, either by increasingthe oxygen transfer rate when the RQ is too high or decreasing theoxygen transfer rate when the RQ is too low or the fermentable sugarfeed rate is adjusted, either by increasing the fermentable sugar feedrate when the RQ is too low or decreasing the fermentable sugar feedrate when the RQ is too high or the fermentable sugar feed rate is alsoadjusted, either by increasing the fermentable sugar feed rate when theRQ is too low and decreasing the fermentable sugar feed rate when the RQis too high or is performed by modulating agitation rate of the culture.17. (canceled)
 18. (canceled)
 19. (canceled)
 20. The method of claim 1wherein the DNA segment encodes an antibody heavy chain or an antibodylight chain, or a fragment of an antibody or each yeast cell comprises aDNA segment encoding a heavy chain polypeptide and a DNA segmentencoding a light chain polypeptide of an antibody.
 21. The method ofclaim 1 wherein the polypeptide is harvested from the culture medium.22. (canceled)
 23. A method for producing an antibody comprising twoheavy chains and two light chains or an antibody fragment in Pichiayeast cells, comprising the steps of: culturing under hypoxic, fed-batchfermentation conditions a population of Pichia yeast cells in a culturemedium, wherein each yeast cell comprises a DNA segment encoding a heavychain polypeptide and DNA segment encoding a light chain polypeptide ofan antibody, wherein said DNA segments are operably linked to aglyceraldehyde-3-phosphate (GAP) transcription promoter and atranscription terminator; harvesting the yeast cells from the culturemedium; and recovering the antibody produced by the yeast cells from theyeast cell-depleted culture medium, wherein the Pichia yeast cells arepreferably selected from the group consisting of Pichia pastoris, Pichiamethanolica, Pichia angusta, and Pichia thermomethanolica. 24.(canceled)
 25. (canceled)
 26. A large scale fermentation processcomprising the steps of: i. culturing yeast cells under large-scale,fed-batch fermentation conditions, wherein said cultured yeast cells areengineered to express a recombinant protein; ii. periodically orcontinuously monitoring the RQ values during the fed-batch fermentation;and determining whether the RQ value falls within a specified range;iii. adjusting at least one culture parameter at least once during thefed-batch fermentation so as to adjust or maintain the RQ value of thefed-batch yeast culture whereby it is within the specified range; andiv. harvesting the yeast cells or the culture medium and recovering therecombinant protein from the harvested cells or culture medium of step(iii), wherein the protein preferably is secreted and/or is an antibodyprotein or antibody fragment.
 27. (canceled)
 28. (canceled) 29.(canceled)
 30. The method of claim 26 wherein the adjusted cultureparameters include one or more of (a) air flow rate, b) oxygenconcentration, (c) feed composition, (d) feed rate, (e) cell density,and (f) agitation or the adjusted culture parameters include one or moreof (a) air flow rate, (b) oxygen concentration, (c) feed composition,(d) feed rate, (e) cell density, and (f) agitation or the adjustedculture parameter comprises one or more of (a) the feed composition, (b)the feed rate and (c) the oxygen transfer rate; and wherein the cultureparameter is adjusted at least once during the process so as to adjustor maintain the RQ value of the fed-batch yeast culture such that it iswithin the specified range or the adjusted culture parameter compriseone or more of (a) the feed composition, (b) the feed rate or (c) theoxygen transfer rate, and is adjusted at least once during the processso as to adjust or maintain the RQ value of the fed-batch yeast culturesuch that it is within the specified range.
 31. (canceled) 32.(canceled)
 33. (canceled)
 34. The process of claim 26 which furtherincludes a batch fermentation preceding the fed-batch fermentationculture or the fed-batch fermentation is conducted for at least (50hours) or the fed-batch fermentation is conducted for at least (70hours).
 35. (canceled)
 36. (canceled)
 37. (canceled)
 38. (canceled) 39.(canceled)
 40. The process of claim 26 wherein the yeast cells arePichia pastoris, polyploid, haploid or diploid.
 41. (canceled) 42.(canceled)
 43. (canceled)
 44. (canceled)
 45. (canceled)
 46. (canceled)47. (canceled)
 48. The process of claim 26 wherein the cell density ofthe fed-batch culture comprises from 1 to 700 g/l wet cell weight. 49.(canceled)
 50. The process of claim 26 wherein in step (iii) the feedrate is increased or decreased so as to adjust the RQ value to fallwithin the specified range or the feed composition is altered byaltering the amount of at least one fermentable sugar or otherhydrocarbon so as to adjust the RQ value to fall within the specifiedrange or the amount of oxygen in the fed-batch fermentation is increasedor decreased during the fed-batch fermentation so as to adjust the RQvalue to fall within the specified range or the amount of at least onefermentable sugar or other fermentable hydrocarbon is increased ordecreased so as to adjust the RQ value to fall within the specifiedrange, the feed rate is increased or decreased during the fed-batchfermentation so as to adjust the RQ value to fall within the specifiedrange.
 51. (canceled)
 52. (canceled)
 53. (canceled)
 54. (canceled) 55.(canceled)
 56. (canceled)
 57. (canceled)
 58. (canceled)
 59. (canceled)60. The process of claim 26 wherein the process results in animprovement in a property selected from antibody purity and antibodyproduction relative to a fed-batch process which does not include step(iii).
 61. The process of claim 26 wherein the specified range for theRQ value in step (iii) is between about 1.08-1.35 or the specified rangefor the RQ value in step (iii) is between about 1.08-1.2.
 62. (canceled)63. (canceled)
 64. (canceled)